1. Field of the Invention
This invention relates to genetic engineering and specifically fusion expression of G-protein coupled receptors (GPCRs).
2. Description of Related Art
G-protein coupled receptors (GPCRs) are a large family of transmembrane protein receptors. All GPRCs share a common structural feature, that is, an extracellular N-terminus, followed by seven transmembrane α-helices connected by three intracellular and three extracellular loops and finally an intracellular C-terminus.
The extracellular regions often have glycosylated residues. The C-terminus and the intracellular loop between the fifth and sixth transmembrane helical regions together form the G protein binding site. There are 800 known GPCRs so far, which can be classified into six classes: Class A (or 1) (Rhodopsin-like); Class B (or 2) (Secretin receptor family); Class C (or 3) (Metabotropic glutamate/pheromone); Class D (or 4) (Fungal mating pheromone receptors); Class E (or 5) (Cyclic AMP receptors); and Class F (or 6) (Frizzled/Smoothened). [Friedricksson et al., Mol. Pharmacol. 63 (6):1256-1272, 2003: and Friedricksson et al., Mol. Pharmacol. 67 (5):1414-1425, 2005]. There is little nucleotide sequence homology between the GPCRs classes.
By coupling with different G proteins, the various GPCRs react to a vast array of extracellular signals, leading to a series of physiology effects including neural transmission, smell, taste, vision and cellular metabolism, differentiation, reproduction and endocrine responses.
Numerous diseases are known to be associated with GPCRs. More than 40% of modern drugs, and over half of the thousands of drugs on the market target GPCRs. These GPCR-targeting drugs are effective treatments of pain, cognizance impairment, high blood pressure, ulcer, nasal inflammation and asthma. Due to the important physiological roles of GPCRs, their structure and function have been intensively studied.
However, wild type GPCRs are unstable in vitro and it is difficult to obtain pure and stable form. Recently, several research groups reported methods used to improve the stability of GPCRs, including (1) insertion of an E. coli T4 phage lysozyme T4L between the ICL3 (intracellular loop 3) and the N-terminus. This approach has been successfully applied to the studies of A2a receptor, CXCR4 receptor, beta-2 adrenergic receptor, D3 dopamine receptor, S1P1 receptor etc. The modification of GPCR with T4L has led to high expression and high yield, and eventually to a high-resolution crystal structure. [Rasmussen et al., Crystal structure of the human beta2 adrenergic G-protein-coupled receptor, Nature 450: 383-387, 2007; Wu et al., Structures of the CXCR4 chemokine GPCR with small-molecule and cyclic peptide antagonists Science 330: 1066-1071, 2010; Chien et al., Structure of the human dopamine D3 receptor in complex with a D2/D3 selective antagonist, Science 330: 1091-1095, 2010; Xu et al., Structure of an agonist-bound human A2A adenosine receptor, Science 332: 322-327, 2011; Hanson et al., Crystal structure of a lipid G protein-coupled receptor; Science 335: 851-855, 2012; and Zou et al., N-terminal T4 lysozyme fusion facilitates crystallization of a G protein coupled receptor Plos One 7: e46039-e46039 2012]. (2) Insertion of bacterial Bril protein in the N-terminus or ICL3. This has been successfully applied to GPCRs such as adenosine A2a receptor, Nociceptin/orphanin FQ receptor, 5HT1b, 5HT2b and SMO receptor, leading to successful determination of their crystal structures. [Liu, W. et al. Structural basis for allosteric regulation of GPCRs by sodium ions, Science 337: 232-236, 2012. Thompson, A. A. et al. Structure of the nociceptiniorphanin FQ receptor in complex with a peptide mimetic Nature 485: 395-399, 2012. Wang, C. et al. Structural Basis for Molecular Recognition at Serotonin Receptors Science 2013. Wang, C. Structure of the human smoothened 7TM receptor in complex with an antitumor agent Nature 2013]. (3) Mutation screening of GPCRs for mutants which possess improved stability with unaffected protein structure and function. This approach has been successfully demonstrated the stable preparation of A2a and beta-1 adrenergic receptor with high yield and high resolution crystal structures [Lebon, G. et al. Agonist-bound adenosine A2A receptor structures reveal common features of GPCR activation. Nature 474: 521, 2011. Warne, A. et al. Structure of a beta1-adrenergic G-protein-coupled receptor Nature 454: 486, 2008]. (4) Using antibody to stabilize the configuration of GPCRs. Using this approach, the Brian Kobilka Laboratory of Stanford University obtained a high resolution crystal structure of beta-2 adrenergic receptor [Bokoch M. P. et al., Ligand-specific regulation of the extracellular surface of a G-protein-coupled receptor, Nature 463: 108-112, 2010].
So far, all proteins used as fusion partners to stabilize GPCRs are prokaryotic proteins, and there has been no report of using a eukaryotic fusion protein partner. Use of eukaryotic fusion protein partners for GPCR protein expression may be advantageous since all GPCRs are present in eukaryotic cells. Therefore it would be desirable to find eukaryotic protein partners for GPCRs fusion expression. Furthermore, even though T4L and Bril proteins have successfully been applied to some GPCRs for expression and purification, they are not useful for many other GPCRs. Thus, additional fusion protein partners are highly desirable.